The long term goal of this project is to understand the structure, function, and regulation of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD), an enzyme that specifically cleaves GPIs, is secreted by pancreatic islets, and circulates in blood associated with high density lipoproteins. GPI-anchored proteins regulate inflammation, are involved in development and signal transduction, play a role in slow viral disease, and comprise the majority of protozoan parasite cell surface antigens. Cleavage and release of GPI-anchored proteins is an important biochemical mechanism for regulating the cell surface expression of these proteins. These proposed studies are aimed at defining the role of apolipoprotein AI, the major protein in high density lipoproteins, in regulating GPI-PLD. Preliminary studies have shown that GPI-PLD associates primarily with lipoprotein particles that contain apolipoprotein AI but not AII in human plasma and that plasma GPI-PLD activity is increased in insulin dependent diabetes mellitus and is regulated by insulin. The specific aims are to: l) isolate GPI-PLD complexes from human plasma using immunoaffinity chromatography to determine the protein and lipid composition, 2) determine if GPI-PLD binds specifically to apolipoprotein AI compared to other apolipoproteins and if altering the conformation of apolipoprotein AI changes binding to GPI-PLD, 3) assess the effect of apolipoprotein AI on GPI-PLD mediated cleavage of GPI-anchored proteins in human skin fibroblasts, and 4) determine if GPI- PLD enhances apolipoprotein AI removal of cellular cholesterol and phospholipids from human skin fibroblasts. Thus, understanding apolipoprotein AI regulation of GPI-PLD has broad implications in a number of vital biological functions with potential alterations in diabetes mellitus and dyslipidemias.